Domain Closure in the Catalytic Chains of Escherichia coli Aspartate Transcarbamoylase Influences the Kinetic Mechanism

Ley, Brenda W.; Kantrowitz, Evan R.; O’Leary, Marion H.; Wedler, Frederick C.

doi:10.1074/jbc.270.26.15620

Cited by 13 publications

(11 citation statements)

References 32 publications

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“…The maximal activity of the C47A/A241C holoenzyme was greater under non-reducing versus reducing conditions (25.6 versus 21 mmol⅐h Ϫ1 ⅐mg Ϫ1 respectively), suggesting that the switch from the T to R state is somewhat rate-limiting in the holoenzyme. This is supported by work done on the wildtype enzyme and D236A, another enzyme with enhanced maximal activity, which suggested that domain closure or "compression," the final step in the T to R transition, is rate-limiting (37,38). There was a significant difference in the nucleotide saturation curves under reducing and non-reducing conditions corresponding to a wild-type-like and R state enzyme, respectively.…”

Section: Discussionsupporting

confidence: 55%

Stabilization of the R Allosteric Structure of Escherichia coli Aspartate Transcarbamoylase by Disulfide Bond Formation

West

Tsuruta

Kantrowitz

2002

Journal of Biological Chemistry

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Here we report the first use of disulfide bond formation to stabilize the R allosteric structure of Escherichia coli aspartate transcarbamoylase. In the R allosteric state, residues in the 240s loop from two catalytic chains of different subunits are close together, whereas in the T allosteric state they are far apart. By substitution of Ala-241 in the 240s loop of the catalytic chain with cysteine, a disulfide bond was formed between two catalytic chains of different subunits. The cross-linked enzyme did not exhibit cooperativity for aspartate. The maximal velocity was increased, and the concentration of aspartate required to obtain onehalf the maximal velocity, [Asp] 0.5 , was reduced substantially. Furthermore, the allosteric effectors ATP and CTP did not alter the activity of the cross-linked enzyme. When the disulfide bonds were reduced by the addition of 1,4-dithio-DL-threitol the resulting enzyme had kinetic parameters very similar to those observed for the wild-type enzyme and regained the ability to be activated by ATP and inhibited by CTP. Small-angle x-ray scattering was used to verify that the cross-linked enzyme was structurally locked in the R state and that this enzyme after reduction with 1,4-dithio-DL-threitol could undergo an allosteric transition similar to that of the wild-type enzyme. The complete abolition of homotropic and heterotropic regulation from stabilizing the 240s loop in its closed position in the R state, which forms the catalytically competent active site, demonstrates the significance that the quaternary structural change and closure of the 240s loop has in the functional mechanism of aspartate transcarbamoylase.Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2) is one of the best characterized allosteric enzymes, so it serves an important role in the study of allosteric regulation. This enzyme catalyzes the committed step of pyrimidine biosynthesis, the carbamoylation of the amino group of L-aspartate by carbamoyl phosphate to form N-carbamoyl-L-aspartate (1). The enzyme demonstrates homotropic cooperativity for the substrate Laspartate and is heterotropically regulated by effectors ATP, CTP (1), and UTP in the presence of CTP (2). Aspartate transcarbamoylase from E. coli is a dodecamer composed of six catalytic chains organized as two trimeric subunits and six regulatory chains organized as three dimeric subunits. Because aspartate transcarbamoylase is so well characterized and serves as a model for allosteric enzymes it is of particular interest to thoroughly understand the molecular mechanism of allostery of this enzyme.Two different structural and functional states of aspartate transcarbamoylase have been characterized (3-6). The low activity, low affinity conformation of the enzyme is called the T state, and the high activity, high affinity conformation of the enzyme is called the R state. The conversion from the T to the R state occurs upon aspartate binding to the holoenzyme in the presence of carbamoyl phosphate, which is the source of the observed homotropic co...

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Section: Discussionsupporting

confidence: 55%

Stabilization of the R Allosteric Structure of Escherichia coli Aspartate Transcarbamoylase by Disulfide Bond Formation

West

Tsuruta

Kantrowitz

2002

Journal of Biological Chemistry

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“…First, the formation of the high activity high affinity active site, which has ordered substrate binding, requires these interdomain bridging interactions. Without the interdomain bridging interactions, not only does the enzyme lose considerable activity and substrate affinity, but it also exhibits a random rather than an ordered mechanism (11). Second, without these interdomain bridging interactions the enzyme cannot be converted into the R structural state except by the bisubstrate analog, PALA (12).…”

Section: Discussionmentioning

confidence: 99%

“…The kinetic data suggest that the elimination of these interactions prevents domain closure, and, therefore, the high affinity, high activity active site cannot form. Furthermore, in the absence of domain closure, the preferred order substrate binding mechanism is no longer observed; rather the mechanism becomes random (11).…”

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confidence: 99%

Domain Bridging Interactions

Sakash¹,

Williams²,

Tsuruta³

et al. 2001

Journal of Biological Chemistry

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Aspartate transcarbamoylase undergoes a domain closure in the catalytic chains upon binding of the substrates that initiates the allosteric transition. Interdomain bridging interactions between Glu 50 and both Arg 167 and Arg 234 have been shown to be critical for stabilization of the R state. A hybrid version of the enzyme has been generated in vitro containing one wildtype catalytic subunit, one catalytic subunit in which Glu 50 in each catalytic chain has been replaced by Ala (E50A), and wild-type regulatory subunits. Thus, the hybrid enzyme has one catalytic subunit capable of domain closure and one catalytic subunit incapable of domain closure. The hybrid does not behave as a simple mixture of the constituent subunits; it exhibits lower catalytic activity and higher aspartate affinity than would be expected. As opposed to the wild-type enzyme, the hybrid is inhibited allosterically by CTP at saturating substrate concentrations. As opposed to the E50A holoenzyme, the hybrid is not allosterically activated by ATP at saturating substrate concentrations. Small angle x-ray scattering showed that three of the six interdomain bridging interactions in the hybrid is sufficient to cause the global structural change to the R state, establishing the critical nature of these interactions for the allosteric transition of aspartate transcarbamoylase.Escherichia coli aspartate transcarbamoylase catalyzes the committed step of pyrimidine biosynthesis, the condensation of carbamoyl phosphate and L-aspartate to form N-carbamoyl-Laspartate (1). The enzyme shows homotropic cooperativity for the substrate L-aspartate and is heterotropically regulated by ATP, CTP (1), and UTP in the presence of CTP (2). The holoenzyme is composed of six catalytic chains (M r 34,000), associated as two trimeric subunits, and six regulatory chains (M r 17,000), associated as three dimeric subunits. The active sites, three/ trimer, are located at the interface between catalytic chains, whereas the allosteric sites, two/dimer, are located on the Nterminal domain of the regulatory chains.Two functionally and structurally different states of aspartate transcarbamoylase have been characterized. The low affinity, low activity conformation of the enzyme is described as the "tense" or T state, whereas the high affinity, high activity conformation of the enzyme is described as the "relaxed" or R state. The conversion from the T state to the R state occurs upon aspartate binding to the enzyme in the presence of carbamoyl phosphate. Structurally, the enzyme elongates by at least 11 Å along the 3-fold axis, and the catalytic trimers and regulatory dimers rotate along their axes of symmetry, 10°and 15°, respectively (3). The allosteric transition can also be monitored in solution using small angle x-ray scattering (SAXS) 1 (4). In addition to the quaternary changes, several tertiary changes also occur because of a rearrangement of several important inter-and intrasubunit interactions. One important intrasubunit interaction that stabilizes the R state is between...

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“…Other mutant enzymes, however, are stabilized in the T state. The Glu50cAla enzyme is unable to close the C chain domains and thus does not exist in the R state conformation (58,71). A Thr82rAla enzyme is structurally fixed in an extreme T state with the T-R equilibrium shifted toward the T state (108).…”

Section: Atcasementioning

confidence: 99%

Allosteric Regulation of Catalytic Activity: Escherichia coli Aspartate Transcarbamoylase versus Yeast Chorismate Mutase

Helmstaedt

Krappmann

Braus

2001

Microbiol Mol Biol Rev

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Allosteric regulation of key metabolic enzymes is a fascinating field to study the structure-function relationship of induced conformational changes of proteins. In this review we compare the principles of allosteric transitions of the complex classical model aspartate transcarbamoylase (ATCase) from Escherichia coli, consisting of 12 polypeptides, and the less complicated chorismate mutase derived from baker's yeast, which functions as a homodimer. Chorismate mutase presumably represents the minimal oligomerization state of a cooperative enzyme which still can be either activated or inhibited by different heterotropic effectors. Detailed knowledge of the number of possible quaternary states and a description of molecular triggers for conformational changes of model enzymes such as ATCase and chorismate mutase shed more and more light on allostery as an important regulatory mechanism of any living cell. The comparison of wild-type and engineered mutant enzymes reveals that current textbook models for regulation do not cover the entire picture needed to describe the function of these enzymes in detail

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Domain Closure in the Catalytic Chains of Escherichia coli Aspartate Transcarbamoylase Influences the Kinetic Mechanism

Cited by 13 publications

References 32 publications

Stabilization of the R Allosteric Structure of Escherichia coli Aspartate Transcarbamoylase by Disulfide Bond Formation

Stabilization of the R Allosteric Structure of Escherichia coli Aspartate Transcarbamoylase by Disulfide Bond Formation

Domain Bridging Interactions

Allosteric Regulation of Catalytic Activity: Escherichia coli Aspartate Transcarbamoylase versus Yeast Chorismate Mutase

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